Our approach to dissecting the processes of endocytosis, glycopprotein biosynthesis and sorting is through isolation and analysis of mutants. We have found that the majority of CHO cell endocytosis mutants, whether non-conditional or temperature-sensitive, isolated by ourselves and others using disparate strategies, represent only two complementation groups. Using our standard isolation procedures we have recently isolated endocytosis mutants of LTk- cells; phenotypes of these mutants differ markedly from the CHO mutants. Acid hydrolases are transported to lysosomes by both an endocytic and an intracellular pathway; the acidic endosome has been proposed as the junction of these pathways, with receptor redistributing from this compartment to both pathways. However, on return of temperature-sensitive CHO cell endocytosis mutants to the permissive temperature, we observe drastically different rates for restoration of surface receptor activity versus intracellular transport. Our data suggest that these pathways meet only after segregation of receptor from the acid hydrolases. We have characterized a glycosylation mutant that 1. transfers oligosaccharide from lipid to protein in decreased amounts; 2. fails to elongate the lipid-linked GlcNAcMan5 intermediate in vivo; 3. accumulates the sugar donor mannosyl phosphoryl dolichol in elevated amounts; 4. elongates the Man5 in vitro in the presence of detergent. This phenotype is consistent with a defect in translocation of the lipid-linked Man5 intermediate from the cytoplasmic to the luminal face of the ER membrane.